Synthesis and early development of hexadecyloxypropylcidofovir: an oral antipoxvirus nucleoside phosphonate

Hexadecyloxypropyl-cidofovir (HDP-CDV) is a novel ether lipid conjugate of (S)-1-(3-hydroxy-2-phosphonoylmethoxypropyl)-cytosine (CDV) which exhibits a remarkable increase in antiviral activity against orthopoxviruses compared with CDV. In contrast to CDV, HDP-CDV is orally active and lacks the nephrotoxicity of CDV itself. Increased oral bioavailability and increased cellular uptake is facilitated by the lipid portion of the molecule which is responsible for the improved activity profile. The lipid portion of HDP-CDV is cleaved in the cell, releasing CDV which is converted to CDV diphosphate, the active metabolite. HDP-CDV is a highly effective agent against a variety of orthopoxvirus infections in animal models of disease including vaccinia, cowpox, rabbitpox and ectromelia. Its activity was recently demonstrated in a case of human disseminated vaccinia infection after it was added to a multiple drug regimen. In addition to the activity against orthopoxviruses, HDP-CDV (CMX001) is active against all double stranded DNA viruses including CMV, HSV-1, HSV-2, EBV, adenovirus, BK virus, orf, JC, and papilloma viruses, and is under clinical evaluation as a treatment for human infections with these agents.     

Replication of human cytomegalovirus in severe combined immunodeficient mice implanted with human retinal tissue.

Because human cytomegalovirus (HCMV) infection and replication are limited to human cells, few animal models can be used to specifically examine the biology of HCMV in vivo. In these studies, fetal human retinal tissue was implanted into the anterior chamber of the severe combined immunodeficient (SCID) mouse eye and subsequently was inoculated with HCMV. Viral replication, localized to glial cells in the xenografts, was first detected 7 days after infection. Thereafter, HCMV replication increased to peak levels through days 21-28 and then gradually decreased to undetectable levels by 8 weeks after infection. The clinical isolate Toledo replicated to higher titers than did strain AD169 or Towne. A comparison of implant age indicated that older tissue could support higher levels of HCMV replication than could younger implants. SCID mice implanted with human retinal tissue provide an excellent model for evaluation of HCMV infection of an ocular structure in vivo.     

Ether lipid-ester prodrugs of acyclic nucleoside phosphonates: activity against adenovirus replication in vitro

The acyclic nucleoside phosphonate cidofovir (CDV) and its closely related analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-adenine ([S]-HPMPA) have been reported to have activity against many adenovirus (AdV) serotypes. A new series of orally active ether lipid-ester prodrugs of CDV and of (S)-HPMPA that have slight differences in the structure of their lipid esters were evaluated, in tissue-culture cells, for activity against 5 AdV serotypes. The results indicated that, against several AdV serotypes, the most active compounds were 15-2500-fold more active than the unmodified parent compounds and should be evaluated further for their potential to treat AdV infections in humans.     

Development of CMX001 for the Treatment of Poxvirus Infections

CMX001 (phosphonic acid, [[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl] ester) is a lipid conjugate of the acyclic nucleotide phosphonate, cidofovir (CDV). CMX001 is currently in Phase II clinical trials for the prophylaxis of human cytomegalovirus infection and under development using the Animal Rule for smallpox infection. It has proven effective in reduction of morbidity and mortality in animal models of human smallpox, even after the onset of lesions and other clinical signs of disease. CMX001 and CDV are active against all five families of double-stranded DNA (dsDNA) viruses that cause human morbidity and mortality, including orthopoxviruses such as variola virus, the cause of human smallpox. However, the clinical utility of CDV is limited by the requirement for intravenous dosing and a high incidence of acute kidney toxicity. The risk of nephrotoxicity necessitates pre-hydration and probenecid administration in a health care facility, further complicating high volume CDV use in an emergency situation. Compared with CDV, CMX001 has a number of advantages for treatment of smallpox in an emergency including greater potency in vitro against all dsDNA viruses that cause human disease, a high genetic barrier to resistance, convenient oral administration as a tablet or liquid, and no evidence to date of nephrotoxicity in either animals or humans. The apparent lack of nephrotoxicity observed with CMX001 in vivo is because it is not a substrate for the human organic anion transporters that actively secrete CDV into kidney cells. The ability to test the safety and efficacy of CMX001 in patients with life-threatening dsDNA virus infections which share many basic traits with variola is a major advantage in the development of this antiviral for a smallpox indication.     

Upregulation of human cytomegalovirus by HIV type 1 in human lymphoid tissue ex vivo

HIV-1 copathogens are believed to play a critical role in progression to AIDS. Human cytomegalovirus (HCMV) has a high prevalence in the general population and is a common copathogen in HIV-1-infected individuals. Important events in copathogen interactions with HIV-1 take place in lymphoid tissue where critical events in HIV-1 disease occur. Here, we used an experimental system of human lymphoid tissue ex vivo to investigate interactions of HCMV with HIV-1. We inoculated ex vivo blocks of human lymphoid tissue with a recombinant strain of HCMV, expressing the green fluorescent protein, and HIV-1 and monitored viral replication and the phenotype of productively infected cells. HCMV readily replicated in tissue blocks as revealed by the release of HCMV viral DNA and an increasing number of viral-positive cells. Immunophenotyping of HCMV-infected cells showed a preferential infection of activated lymphocytes. The number of these cells significantly increased in HIV-1-coinfected tissues. Accordingly, HCMV replication was enhanced 2- to-3 fold. This upregulation occurred in tissues infected with either CXCR4- or CCR5-utilizing HIV-1. Thus, HIV-1 creates new targets for HCMV, which may explain the strong association of HCMV with HIV-1 infection in vivo. Ex vivo-infected human lymphoid tissue constitutes a model to study the mechanisms of HCMV tissue pathogenesis and its interactions with HIV-1 and this model may provide new targets for anti-HIV-1 therapy.     

Efficacy of CMX001 as a prophylactic and presymptomatic antiviral agent in New Zealand white rabbits infected with rabbitpox virus, a model for orthopoxvirus infections of humans

CMX001, a lipophilic nucleotide analog formed by covalently linking 3-(hexdecyloxy)propan-1-ol to cidofovir (CDV), is being developed as a treatment for smallpox. CMX001 has dramatically increased potency versus CDV against all dsDNA viruses and, in contrast to CDV, is orally available and has shown no evidence of nephrotoxicity in healthy volunteers or severely ill transplant patients to date. Although smallpox has been eliminated from the environment, treatments are urgently being sought due to the risk of smallpox being used as a bioterrorism agent and for monkeypox virus, a zoonotic disease of Africa, and adverse reactions to smallpox virus vaccinations. In the absence of human cases of smallpox, new treatments must be tested for efficacy in animal models. Here we first review and discuss the rabbitpox virus (RPV) infection of New Zealand White rabbits as a model for smallpox to test the efficacy of CMX001 as a prophylactic and early disease antiviral. Our results should also be applicable to monkeypox virus infections and for treatment of adverse reactions to smallpox vaccination.     

A pilot study of cidofovir in patients with kaposi sarcoma

A clinical trial was conducted to test the activity of cidofovir (CDV), a drug with in vitro activity against Kaposi sarcoma (KS)-associated herpesvirus (KSHV), in KS. Five patients with human immunodeficiency virus-associated KS (4 receiving antiretroviral therapy) and 2 patients with classical KS were administered CDV (5 mg/kg/dose) weekly for 2 weeks and then every other week. All 7 patients had progression of their KS at a median of 8.1 weeks (range, 5-27 weeks). Skin biopsy specimens of KS lesions showed no change in expression of latent or early lytic genes, but, in the 1 assessable patient, there was decreased expression of a late lytic gene. There was no decrease in the virus load of KSHV in peripheral blood mononuclear cells. This study does not provide proof of principle for the treatment of KS with CDV. However, it remains possible that antiherpesvirus therapy can be developed for herpes-induced tumors.     

Plecanatide and dolcanatide,novel guanylate cyclase-C agonists,ameliorate gastrointestinal inflammation in experimental models of murine colitis

AIM: To evaluate the effect of orally administeredplecanatide or dolcanatide, analogs of uroguanylin, on amelioration of colitis in murine models.METHODS: The cyclic guanosine monophosphate(cG MP) stimulatory potency of plecanatide and dolcanatide was measured using a human colon carcinoma T84 cellbased assay. For animal studies all test agents were formulated in phosphate buffered saline. Sulfasalazine or 5-amino salicylic acid(5-ASA) served as positive controls. Effect of oral treatment with test agents on amelioration of acute colitis induced either by dextran sulfate sodium(DSS) in drinking water or by rectal instillation of trinitrobenzene sulfonic(TNBS) acid, was examined in BALB/c and/or BDF1 mice. Additionally, the effect of orally administered plecanatide on the spontaneous colitis in T-cell receptor alpha knockout(TCRα-/-) mice was also examined. Amelioration of colitis was assessed by monitoring severity of colitis, disease activity index and by histopathology. Frozen colon tissues were used to measure myeloperoxidase activity.RESULTS: Plecanatide and dolcanatide are structurally related analogs of uroguanylin, which is an endogenous ligand of guanylate cyclase-C(GC-C). As expected from the agonists of GC-C, both plecanatide and dolcanatide exhibited potent cG MP-stimulatory activity in T84 cells. Once-daily treatment by oral gavage with either of these analogs(0.05-0.5 mg/kg) ameliorated colitis in both DSS and TNBS-induced models of acute colitis, as assessed by body weight, reduction in colitis severity(P 0.05) and disease activity index(P 0.05). Amelioration of colitis by either of the drug candidates was comparable to that achieved by orally administered sulfasalazine or 5-ASA. Plecanatide also effectively ameliorated colitis in TCRα-/- mice, a model of spontaneous colitis. As dolcanatide exhibited higher resistance to proteolysis in simulated gastric and intestinal juices, it was selected for further studies. CONCLUSION: This is the first-ever study reporting the therapeutic utility of GC-C agonists as a new class of orally delivered and mucosally active drug candidates for the treatment of inflammatory bowel diseases.     

巨细胞病毒感染与动脉粥样硬化关系的研究

近年,动脉粥样硬化(Atherosclerosis,As)发生的病毒病因学研究日益受到人们的关注.本研究通过应用ELISA、PCR及原位杂交等技术对45例As病人及22例正常者血清及血管组织配对标本检测了HCMV—sIgG、sIgM及HCMV IE、L基因片段,以及其在血管组织中的分布.结果显示:As组中HCMV—sIgG抗体水平显著高于对照组,sIgM仅见于As组.HCMV IE及L基因的阳性率也显著高于对照,并且发现HCMV DNA多见于内膜下肌层及中膜平滑肌的细胞核,强烈提示HCMV感染与As的发生密切相关.同时,本文还就HCMV—sIgG及sIgM与HCMV基因检出的关系及意义作了探讨,为As的临床早期诊断、早期预防及针对性治疗奠定了基础.     

Comparison of benzimidazole nucleosides and ganciclovir on the in vitro proliferation and colony formation of human bone marrow progenitor cells

Recently we have shown that certain benzimidazole ribonucleosides are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. Because antiviral drugs used to treat HCMV and human immunodeficiency virus (HIV) infections can suppress marrow progenitors, we have evaluated the most promising of the new benzimidazoles for their effects on human bone marrow cells in vitro. In an initial study of the bone marrow toxicity of one of the most active compounds, 100 μm 2-bromo-5,6-dichloro-1-(β-d -ribofuranosyl)-benzimidazole (BDCRB) inhibited cell proliferation by 20% over a 10 d period compared to 52% inhibition by 100 μm ganciclovir, the drug currently most used to treat HCMV infections. The effects of these drugs and selected other benzimidazole nucleosides were evaluated more extensively in haemopoietic progenitor cell colony formation assays. Colony formation was determined at 2 weeks and scored as either burst forming units-erythroid (BFU-E), or colony forming units-granulocyte/macrophage (CFU-GM). At the highest concentration tested, 100 μm BDCRB only moderately affected BFU-E or CFU-GM formation (31% and 47% inhibition, respectively). This concentration is 10-fold higher than that required to produce a 10 000-fold reduction in virus titre. Evaluation of the 2-chloro analog of BDCRB (TCRB) which is less potent against HCMV, its 5′-deoxy analog (5′-dTCRB) which is more potent, and the 2-unsubstituted compound (DRB) gave the following order of haemopoietic toxicity: DRB > TCRB≥ 5′-dTCRB > BDCRB. In contrast to the benzimidazoles, ganciclovir decreased colony formation by 84% for BFU-E and 86% for CFU-GM at 100 μm . These results establish that certain benzimidazole nucleosides are less toxic to haemopoietic progenitors than the preferred drug now being used clinically for HCMV infections. The results also establish that different structure–activity relationships exist for antiviral activity and progenitor cell toxicity, thereby suggesting that different mechanisms are involved in the two types of drug action.     

Human haematopoietic stem cells that mediate long-term in vivo engraftment are not susceptible to infection by human cytomegalovirus

A human/sheep xenograft model was used to evaluate whether long-term engrafting haematopoietic stem cells (HSC) are susceptible to human cytomegalovirus (HCMV) infection. CD34+ Lin- HSC were isolated by fluorescence-activated cell sorting (FACS) from the bone marrow (BM) of HCMV-positive and HCMV-negative normal donors. Cells from the latter group were infected in vitro with HCMV. HCMV DNA was detected in both cell populations by nested-polymerase chain reaction (PCR) and fluorescence in situ hybridization. Cells were transplanted into separate groups of fetal sheep at concentrations of 1.3-5.0 x 105 cells per fetus. Multilineage human haematopoietic cell engraftment, including CD34+ cells, was detected in the BM and peripheral blood of recipients up to 16 months post-transplant as assessed by FACS analysis and PCR for HLA-DQalpha. Levels of engraftment varied (1.2-24.3%) but no sheep exhibited HCMV-positive cells. To ensure that our inability to detect HCMV-positive cells was not due to immune-elimination of HCMV-infected cells, 3.8-10 x 105 HCMV-positive uncharacterized BM stromal cells were transplanted into fetal sheep. At 5 weeks post-transplant several organs were HLA-DQalpha- and HCMV-positive, confirming that HCMV was detectable. These results provide evidence that the long-term engrafting HSC is not a primary target of HCMV and suggest that HCMV infection of human haematopoietic cells is exercised at the level of committed progenitors.     

Activity of oral drugs against Leishmania tropica in human macrophages in vitro

Because of the need for orally active antileishmanial agents, orally administrable drugs have sometimes been used to treat human leishmaniases without prior demonstration of efficacy in experimental models. The antileishmanial activity of such agents was tested against Leishmania tropica (a cause of cutaneous leishmaniasis) within human macrophages in vitro. Although trimethoprim + sulfamethoxazole and isoniazid + rifampin have been reported as efficacious orally in certain human studies of cutaneous disease, these drugs were ineffective in vitro (less than or equal to 40% parasite elimination) at peak achievable serum levels. The combination of allopurinol and Pentostam is being tested in humans. In vitro, allopurinol (5 micrograms/ml) augmented the antileishmanial effect of a low concentration of Pentostam (5 micrograms/ml) but not of a higher concentration of Pentostam (20 micrograms/ml). Nifurtimox is a nitrofuran which has questionable activity against human cutaneous disease. Nifurtimox was similarly only 50% effective in vitro at peak achievable serum levels (1.0-3.0 micrograms/ml). However, furazolidone, another orally administered nitrofuran, eliminated 92% of parasites at 1.0 micrograms/ml. Chlorpromazine and quinacrine are concentrated in tissues that are susceptible to infection by Leishmania. Chlorpromazine and quinacrine eliminated only 15% and 35% of organisms in vitro at achievable serum levels (less than or equal to 0.3 microgram/ml), but eliminated virtually all organisms in vitro at possible achievable tissue levels. Both the negative and the positive data of this report may aid in selection of effective orally active agents for in vivo trials.     

Disease-modifying activity of SB 273005, an orally active, nonpeptide alphavbeta3 (vitronectin receptor) antagonist, in rat adjuvant-induced arthritis

OBJECTIVE: To evaluate the effects of SB 273005, a potent, orally active nonpeptide antagonist of the integrin avbeta3 vitronectin receptor, on joint integrity in rats with adjuvant-induced arthritis (AIA). METHODS: Male Lewis rats with AIA were orally dosed either prophylactically (days 0-20) or therapeutically (days 10-20) with SB 273005. Efficacy was determined by measurement of paw inflammation, assessment of bone mineral density using dual-energy x-ray absorptiometry (DEXA), magnetic resonance imaging (MRI), and histologic evaluation. RESULTS: SB 273005 is a potent antagonist of the closely related integrins, avbeta3 (Ki = 1.2 nM) and alphavbeta5 (Ki = 0.3 nM). When SB 273005 was administered prophylactically to AIA rats twice per day, it inhibited paw edema at doses of 10, 30, and 60 mg/kg, by 40%, 50%, and 52%, respectively. Therapeutic administration twice daily was also effective, and a reduction in paw edema was observed at 30 mg/kg and 60 mg/kg of the antagonist (by 36% and 48%, respectively). SB 273005 was also effective when administered once per day, both prophylactically and therapeutically. Significant improvement in joint integrity in treated rats was shown using DEXA and MRI analyses. These findings were confirmed histologically, and significant protection of bone, cartilage, and soft tissue was observed within the joint. CONCLUSION: Symptoms of AIA in rats were significantly reduced by either prophylactic or therapeutic treatment with the alphavbeta3 antagonist, SB 273005. Measurements of paw inflammation and of bone, cartilage, and soft tissue structure indicated that this compound exerts a protective effect on joint integrity and thus appears to have disease-modifying properties.     

Detection of human cytomegalovirus in clinical specimens by DNA-DNA hybridization

A diagnostic assay has been developed to detect human cytomegalovirus (HCMV) DNA in clinical specimens with 32P-labeled cloned fragments of HCMV strain AD169. The labeled probe can detect 10 pg of HCMV DNA and fails to hybridize to DNA from other herpesviruses or human DNA. The assay correctly identified 22 (92%) of 24 coded urine specimens culture positive for HCMV and 23 (88%) of 26 urine specimens culture negative for HCMV. In a prospective study of 67 buffy-coat specimens from recipients of bone marrow transplants HCMV DNA was detected in 13 (93%) of 14 culture-positive samples. Of 53 buffy-coat specimens culture negative for HCMV, 32 were hybridization negative for HCMV DNA. However, in 20 of 21 buffy-coat specimens positive for HCMV by hybridization but negative by culture there was evidence that the hybridization assay was correct and more sensitive than currently available tissue culture techniques.     

Comparative analysis of a double primer PCR assay with plasma, leukocytes and antigenemia for diagnosis of active human cytomegalovirus infection in bone marrow transplant patients

The aim of the study was to determine the prognostic value of a double primer PCR assay to detect human cytomegalovirus (HCMV) infection or disease in bone marrow transplant (BMT) recipients. A total of 209 blood samples including peripheral blood mononuclear cells (PBMN), polymorphonuclear (PMN) leukocytes and plasma from 26 BMT recipients were tested by PCR assay. To discriminate between latent and active HCMV infection, 177 blood samples were also tested by a quantitative antigenemia assay. HCMV serology status of donors and recipients was determined before transplantation by an enzyme immunosorbent assay method. Using the double primer PCR assay, the number of positive samples increased by an average of 11.6%. Symptomatic active HCMV infection was diagnosed in 14 (53.8%) out of 26 BMT patients. There was a good association between double primer PCR assay of PMN leukocytes and antigenemia assays for detection of active HCMV infection in all patients. Detection of HCMV DNA in PMN leukocytes of BMT patients by double primer PCR assay can be an alternative method for antigenemia assay. However, quantitative PCR methods will be necessary for monitoring antiviral treatment.     

人巨细胞病毒活动性感染患者颈动脉超声检查分析

目的：探讨人巨细胞病毒（HCMV）活动性感染患者的颈动脉超声特点。方法：应用免疫荧光法检测外周血白细胞HCMV-PP65作为HCMV活动性感染的指标，观察56例HCMV活动性感染相关性和171例非HCMV活动性感染相关性动脉粥样硬化患者的颈动脉超声结果，同时比较年龄、高血压、糖尿病、吸烟等血管危险因素。结果：HCMV活动性感染组年龄、高血压、高脂血症、糖尿病、吸烟等危险因素以及内膜增厚和斑块形成的发生率均与非HCMV活动性感染组无显著差异，HCMV活动性感染组颈内动脉同时存在≥2种不同类型斑块的发生率显著高于非HCMV活动性感染组（52．94％比12．5％，P〈0．01）。结论：颈内动脉同时存在≥2种不同类型斑块可能与HCMV活动性感染相关。     

Cidofovir Activity against Poxvirus Infections

Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, HPMPC] is an acyclic nucleoside analog approved since 1996 for clinical use in the treatment of cytomegalovirus (CMV) retinitis in AIDS patients. Cidofovir (CDV) has broad-spectrum activity against DNA viruses, including herpes-, adeno-, polyoma-, papilloma- and poxviruses. Among poxviruses, cidofovir has shown in vitro activity against orthopox [vaccinia, variola (smallpox), cowpox, monkeypox, camelpox, ectromelia], molluscipox [molluscum contagiosum] and parapox [orf] viruses. The anti-poxvirus activity of cidofovir in vivo has been shown in different models of infection when the compound was administered either intraperitoneal, intranasal (aerosolized) or topically. In humans, cidofovir has been successfully used for the treatment of recalcitrant molluscum contagiosum virus and orf virus in immunocompromised patients. CDV remains a reference compound against poxviruses and holds potential for the therapy and short-term prophylaxis of not only orthopox- but also parapox- and molluscipoxvirus infections.     

Human cytomegalovirus (HCMV) infection in paediatric patients given allogeneic bone allogeneic bone marrow transplantation: role of early antiviral treatment for HCMV antigenaemaia on Patients' outcome

Summary. In a prospective study, we evaluated the role of early treatment with ganciclovir of human cytomegalvirus (HCMV) pp65-antigenaemia, as well as the risk factors related to the infection in 48 paediatric patients given an allogeneic bone marrow transplantation (BMT). HCMV infection occurred in 24 children, the overall actuarial risk of infection at 120 d being 51%. Development of acute graft-versus-host disease (GVHD), steroid therapy and serological status of both recipient and donor were the most powerful predictors of HCMV infection, none of the six seronegative patient/donor pairs developing HCMV infection. Considering only the seropositive recipients and patients given a seropositive marrow (42 cases), the actuarial risk of developing HCMV antigenaemia in patients with acute GVHD was 76%v 27% in those with or without GVHD (P < 0.005) and 79%v 15% respectively in patients who did or did not receive steroid therapy (P < 0.001). HCMV disease developed in 5/24 children with pp65-antigen-aemia, which was detected before diagnosis in all cases but one. All patients with pp65-positive cells were treated with ganciclovir at a dose of 5 mg/kg twice daily for 14d. In patients without acute GVHD no maintenance therapy was adminis-tered, whereas children with active acute GVHD were given additional therapy with ganciclovir at a dose of 5 mg/kg/d for 14d. Ganciclovir produced complete clearing of viraemia and antigenaemia, with some patients presenting recurrences of antigenaemia, which were treated according to the above-mentioned schedule. Likewise, HCMV disease completely resolved after treatment with ganciclovir and no patients died from HCMV-related interstitial pneumonia. Our results suggest that an early short-term therapy with ganciclovir after demonstration of antigenaemia can be effective in reducing or abolishing HCMV-related mortality. This approach elim-inates the use of ganciclovir in patients not presenting HCMV reactivation and therefore not benefiting from therapy. The administration of ganciclovir limited to the period needed to obtain antigenaemia clearance could also have the advantage of reducing myelotoxicity.     

Mechanisms of human cytomegalovirus (HCMV) (re)activation and its impact on organ transplant patients

Human cytomegalovirus (HCMV) infection plays an important role in transplant patients. Its impact is both direct and indirect. This review focuses on new aspects of HCMV (re)activation and HCMV related pathology, particularly HCMV-associated renal allograft injury. During the last two years we have learned that HCMV is more frequently (re)activated, even in healthy people, than previously expected. Inflammatory as well as stress mediators and some drugs may (re)activate the virus by using distinct intracellular pathways. Commonly, HCMV (re)activation is accompanied by HCMV antigenemia/DNAemia, suggesting that precursor cells in the bone marrow play an important role as a reservoir of latent virus. However, local HCMV (re)activation (colon, lung) without detection of active HCMV infection in the peripheral blood is possible. In healthy people a sufficient type 1 T-cell response controls the active HCMV infection, while in patients with severe immune deficiency (AIDS, high-dose immunosuppression) the virus can spread in an uncontrolled fashion and induce ‘classic’ HCMV disease. In patients with moderate immune deficiency (e.g. long-term transplant patients on low-dose immunosuppression) virus spreading is controlled but the elimination of cells harboring the active virus may be insufficient. The resulting persistent HCMV antigenemia may induce chronic inflammatory processes leading to tissue injury, particularly in the allograft. Therefore, antiviral therapy may be useful in patients suffering from graft deterioration with otherwise clinically symptomless HCMV infection. HCMV-related immune deficiency with an increased risk of developing bacterial/fungal superinfections is frequently seen in patients with symptomatic HCMV disease but not in asymptomatic CMV antigenemia. The risk of developing superinfections can be predicted by flow-cytometric monitoring of peripheral blood monocytes.     

Maternal administration of busulfan before in utero transplantation of human hematopoietic stem cells enhances engraftments in sheep

In utero transplantation (IUT) of human hematopoietic stem cells has been conducted in sheep, which are used as large animal models of human hematopoietic reconstitution and models for clinical IUT; however, the levels of engraftment have generally been low. Busulfan (BU), a myeloablative agent, is often administered to patients before hematopoietic stem cells transplantation to improve the engraftment. In this study, hematopoietic activity was evaluated in adult sheep after administering BU at different doses. Next, pregnant ewes were administered BU, and dams as well as their fetuses were evaluated, as BU readily crosses the sheep placenta. Then, the BU dose with the desired outcomes was selected and administered to pregnant ewes at 2 or 6 days before performing IUT using human cord blood CD34(+) cells. The engraftment was evaluated in recipients that underwent IUT in the presence or absence of BU. As a result, hematopoietic activity was safely and transiently suppressed in adult sheep treated with 5 to 7.5 mg/kg BU. BU crossed the sheep placenta, and fetal sheep were indeed conditioned by administering 3 mg/kg BU to pregnant ewes. Engraftment of human CD34(+) cells in fetal recipients was enhanced when IUT was carried out 6 days post-BU. Up to 3.3% engraftment levels (in terms of bone marrow colony-forming units) were achieved with the IUT of 0.72 to 2.4 million CD34(+) cells when BU was used. BU can be administered to pregnant ewes to effectively condition the fetal recipient for IUT with enhanced engraftment of donor cells.